Method for detecting whether an organism is homozygous or heterozygous using labelled primers and RFLP

ABSTRACT

The invention is a method for detecting whether an organism is homozygous or heterozygous in a target nucleotide. The method combines hybridization of two differently labelled probes A and B on either side of a restriction site, restriction endonuclease digestion, and detection of the labels in the resulting digest. If an organism is: homozygous then the labels will be present in the ratio 1:1 or 1:0; or if heterozygous then the labels will be present in the ratio 1:2. Either immobilized array technology or electrophoretic separation may be used.

FIELD OF THE INVENTION

The present invention relates generally to a method for detecting thepresence or absence of one or more nuclcotides in a target nucleotidesequence. More particularly, the present invention contemplates adiagnostic assay,for the presence or absence of a particular mutation orpolymorphism in a target nucleotide sequence. Even more particularly,the present invention combines differential hybridization or restrictionendonuclease digestion with either immobilized array technology orelectrophoretic separation to detect the presence or absence of amutation or polymorphism in a target nucleotide sequence. The presentinvention further provides a kit to facilitate conducting the diagnosticassay as well as means, and more particularly data processing-assistedmeans, to automate or semi-automate the performance of the diagnosticassay.

BACKGROUND OF THE INVENTION

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any othercountry.

The increasing sophistication of recombinant DNA technology is greatlyfacilitating research and development in the medical, veterinary,agricultural and horticultural industries. This is particularly the casein the area of diagnostics for human disease conditions. As a greaterunderstanding of genomics is achieved and with the completion or nearcompletion of genome sequencing for a range of animals and mammals,including humans, and a range of microorganisms, there will be greateropportunities to develop diagnostic assays for a wide range of geneticbased conditions.

Diagnostic techniques based on nucleic acid hybridization areunparalleled in their ability to identify and quantify genetic materialof particular organisms or groups of genetically related organisms. Theprovision of DNA microfabricated array (micro-array) techniques nowallows an “order of magnitude” increase in speed and specificity forthis kind of gene-based analysis. For example, reference may be made toSouthern (WO 89/10977; U.S. Pat. No. 6,045,270), Chee et al. (U.S. Pat.No. 5,837,832), Cantor et al. (U.S. Pat. No. 6,007,987) and Fodor et al.(U.S. Pat. No. 5,871,928).

Until recently, the nucleic acid probes used in nucleic acidhybridizations were mostly obtained empirically by isolating nucleicacid fragments from targeted organisms or genes. However, it is nowpossible to design and synthesize nucleic acid probes using data fromthe international sequence databases (e.g. the GenBank and EMBLdatabases). These databases of known gene sequences have been increasingtenfold in size every five years for many years and now contain arepresentative sample of most genes and most major groups of organisms.

Generally, DNA micro-arrays use spots of detector oligonucleotides orprobes positioned in arrays on a solid support, typically a glass wafer.The probes are allowed to hybridize with sample nucleic acids, whichcontain the target nucleic acids and which have been fluorescentlylabelled. The probes and target nucleic acids of the sample are allowedto hybridize under conditions that only detect exact or almost exactcomplementarity between the probes and the target nucleic acids. If atarget nucleic acid complements and hybridizes to a particular probe inthe array, the spot will fluoresce. Recording the fluorescence of thespots enables one to assess which target sequences are present in thenucleic acids mixture.

Sequence information, obtained from native RNA or DNA molecules, is usedto determine the sequence of the synthesized oligonucleotide probes andthis information is usually stored in computer databases and manipulatedusing software. Each probe is synthesized so that it containsnucleotides in any order (sequence) that matches a part of a knownnative nucleotide sequence or the complement of a part of that sequence.Oligonucleotide probes used in conventional arrays are typically 10-25nucleotides long.

Currently oligonucleotide probes are most commonly used in micro-arraysto identify and quantify the mRNA transcripts from genes. Thesemicro-arrays usually contain probes representing several differenttarget sequences from each gene sequence and these probes are usuallychosen to be target specific (i.e. they hybridize with just one targetpolynucleotide). Thus, these micro-arrays contain many more probes thanthe number of target polynucleotides they are designed to detect.

Compared to conventional nucleic acid analysis techniques includingrestriction fragment length polymorphism (RFLP) analysis and thepolymerase chain reaction (PCR), DNA micro-arrays provide a facile andrapid means of detecting and measuring the expression of differentgenes. They have also been used to detect variants of well characterizednucleic acid molecules (i.e. to detect genetic polymorphisms andgenotypes). However, despite their promise as tools for diagnosinginfectious diseases as well as genetic disorders, the development ofmicro-arrays for routine diagnosis appears to be slow. This is probablydue to the relatively high cost of designing, developing and producingmicro-arrays that could detect a larger number of targetpolynucleotides. New methods and reagents are, therefore, required torealize this promise and the present invention helps to meet that need.

In accordance with the present invention, the inventors have developedan improved assay system which can readily identify changes innucleotides within a target nucleotide sequence and whether the mutationor polymorphism is present in homozygous or heterozygous form. The assayof the present invention has wide applicability for a range of genetictesting of humans, animals, microorganism and plants. The instant assayhas particular utility in microarray-based assay procedures.Furthermore, many individual subjects can be analyzed on the samemicro-array, which will allow large-scale genetic testing in acost-effective manner.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1, <400>2, etc. A sequence listing isprovided after the claims.

The present invention provides a means for detecting the presence orabsence of a particular nucleotide or group of nucleotides in a targetsequence. The assay comprises the selection or generation of forward andreverse amplification primers which, in one embodiment, are optionallyboth or singularly labelled with reporter molecules capable of providingseparate identifiable signals, i.e. signals which can be distinguishedwith respect to each other. Alternatively, the amplification isconducted using unlabelled primers and detection is accomplished byhybridization of a probe differential labelled at its 5′ and 3′ termini.In one aspect, at least one of the pairs of primers further comprise tagsequences having sense and complementary sequences immobilized to asolid support. In addition, one or both primers may either introduce orremove a restriction endonuclease site within the target sequencedepending on the presence or absence of a mutation sought to bedetected. Alternatively, the primers may differentially hybridize to atarget sequence. Following amplification to incorporate the tagsequence, if present, and reporter molecules and, in one embodiment, toadd or remove a restriction endonuclease site, the amplified product isdigested with the enzyme whose site has been introduced or removed andsingle-stranded forms subjected to immobilization conditions on thesolid support. The presence or absence of the mutation or polymorphismdetermines whether the restriction endonuclease digests the targetsequence and this in turn affects whether or not the reporter moleculeson the respective primers are present on the captured amplifiedproducts. Due to the differential nature of the signals produced by thereporter molecules, a determination can be made as to the presence orabsence of the mutation or polymorphism. One particular form of thisembodiment is shown in FIG. 1. In another embodiment, differentialrestriction endonuclease digestion is assessed electrophoretically. Inthis embodiment, the tag sequence may still be present but is notrequired for electrophoretic separation. In one particular embodiment,amplification primers are employed without any reporter molecules beingattached. In a further embodiment, one or both primers compriseschemically modified bases, nucleotides or phosphate linkages renderingthe strand resistant to exonuclease digestion. This permits thegeneration of single-stranded DNA molecules. The presence or absence ofa restriction endonuclease site is then determined by hybridizing aprobe molecule comprising two different reporter molecules to a regionencompassing the putative restriction site. This partial double-strandedDNA is then subjected to restriction endonuclease digestion and analyzedas above. One form of this particular embodiment is shown in FIG. 4.

Accordingly, one aspect of the present invention contemplates method fordetermining the presence or absence of a homozygous or heterozygouschange in one or more nucleotides within a target nucleotide sequence,said method comprising:—

-   -   amplifying said target nucleotide sequence using forward and        reverse primers to produce an amplified product, wherein at        least one of said primers is labelled with a reporter molecule        capable of facilitating the provision of an identifiable signal        which can be distinguished from another reporter molecule if        both primers are labelled and wherein at least one primer and        its complementary form comprises a complementary sequence to an        oligonucleotide sequence anchored to a solid support and wherein        one or more of said forward or reverse primers introduces,        abolishes or hybridizes to a target site within the amplified        product in the presence or absence of a change in one or more        nucleotides, and    -   subjecting said amplified product to detection means.

Another aspect of the present invention provides a method fordetermining the presence or absence of a homozygous or heterozygouschange in one or more nucleotides within a target nucleotide sequence,said method comprising:

-   -   amplifying said target nucleotide sequence using forward and        reverse primers to produce an amplified product wherein each of        said primers is labelled with a reporter molecule capable of        facilitating the provision of an identifiable signal which can        be distinguished from each other and wherein at least one primer        and its complementary form comprises a complementary sequence to        an oligonucleotide sequence anchored to a solid support and        wherein one or more said forward or reverse primers introduces        or abolishes a restriction endonuclease site within the        amplified product in the presence or absence of a change in one        or more nucleotides;    -   digesting the amplified product with the restriction        endonuclease whose site has been potentially introduced or        abolished in said amplified product and subjecting        single-stranded forms of the amplified product subjected to        hybridization to conditions to permit annealing to a set of said        immobilized oligonucleotides comprising oligonucleotides which        are sense or complementary to a portion of the amplified        sequence introduced by at least one primer; and    -   detecting the relative proportion of signal by the reporter        molecules wherein an equal proportion of different signals or        the substantial presence of only one signal represents a        bomozygous presence or absence of change in the target        nucleotide sequence and wherein the presence of a differential        signal represents a heterozygous presence or absence of said        change in target nucleotide sequence.

In another embodiment, the present invention contemplates a method fordetermining the presence or absence of a homozygous or heterozygouschange in one or more nucleotides within a target nucleotide sequence,said method comprising:—

-   -   amplifying said target nucleotide sequence using forward and        reverse primers to produce an amplified product wherein one        primer comprises one or more chemically modified nucleotides,        bases or phosphodiester bonds such that a nucleotide strand        which extends from said primer is substantially resistant to        exonuclease activity and wherein the other primer comprises a        nucleotide sequences having sense and complementary sequences        immobilized to a solid support and wherein one or more said        forward or reverse primers introduces or abolishes a restriction        endonuclease site within the amplified product in the presence        or absence of a change in one or more nucleotides;    -   digesting the amplified product with an exonuclease to digest        the strand not amplified by the primer comprising the        exonuclease-resistant nucleotides, bases or phosphodiester        linkages to generate a single-stranded nucleic acid molecule        comprising the potential presence or absence of a restriction        endonuclease site and a nucleotide sequence complementary to an        oligonucleotide sequence immobilized to said solid support;    -   hybridizing to said single-stranded nucleic acid molecule a        probe that contains complementarity to the restriction site that        may have been introduced to generate a partial double-stranded        molecule wherein the probe comprises two reporter molecules        capable of facilitating the provision of identifiable signals        which can be distinguished from each other;    -   digesting the partially double-stranded molecule with the        restriction endonuclease whose site has been potentially        introduced or abolished in said amplified product and subjecting        the digested molecule to conditions to permit annealing to a set        of said immobilized oligonucleotides comprising oligonucleotides        which are sense or complementary to a portion of the amplified        sequence introduced by at least one primer; and    -   detecting the relative proportion of signal by the reporter        molecules wherein an equal proportion of different signals or        the substantial presence of only one signal represents a        homozygous presence or absence of change in the target        nucleotide sequence and wherein the presence of a differential        signal represents a heterozygous presence or absence of said        change in target nucleotide sequence.

Another aspect of the present invention contemplates a method fordetermining the presence or absence of a homozygous or heterozygouschange in one or more nucleotides within a target nucleotide sequence,said method comprising:

-   -   amplifying said target nucleotide sequence using forward and        reverse primers to produce an amplified product wherein each of        said primers is labelled with a reporter molecule capable of        facilitating the provision of an identifiable signal which can        be distinguished from each other and wherein one or more of said        forward or reverse primers introduces or abolishes a restriction        endonuclease site within the amplified product in the presence        or absence of a change in one or more nucleotides; and    -   digesting the amplified product with the restriction        endonuclease whose site has been potentially introduced or        abolished in said amplified product and subjecting the amplified        product subjected to digestion to conditions to permit        electrophoretic separation of said digested products wherein the        pattern of electrophoretic separation and/or the pattern of        reporter molecule signaling is indicative of the homozygous        presence or absence or the heterozygous presence or absence of        said change in target sequence.

In a related embodiment, the present invention provides a method fordetermining the presence or absence of a homozygous or heterozygouschange in one or more nucleotides within a target nucleotide sequencesuch as but not limited to, said method comprising:—

-   -   amplifying said target nucleotide sequence using forward and        reverse primers to produce an amplified product wherein one or        more of said forward or reverse primers introduces or abolishes        a restriction endonuclease site within the amplified product in        the presence or absence of a change in one or more nucleotides;        and    -   digesting the amplified product with the restriction        endonuclease whose site has been potentially introduced or        abolished in said amplified product and subjecting the amplified        product subject to digestion to conditions to permit        electrophoretic separation of said digested products wherein the        pattern of electrophoretic separation is indicative of the        homozygous presence or absence or the heterozygous presence or        absence of said change in target sequence.

A further aspect of the present invention provides a method fordetermining the presence or absence of a homozygous or heterozygouschange in one or more nucleotides within a target nucleotide sequence,said method comprising:

-   -   amplifying said target nucleotide sequence using forward and        reverse primers to produce an amplified product wherein each of        said primers is labelled with a reporter molecule capable of        facilitating the provision of an identifiable signal which can        be distinguished from each other and wherein at least one primer        and its complementary form comprises a complementary sequence to        an oligonucleotide sequence anchored to a solid support and        wherein one or more said forward or reverse primers introduces a        restriction endonuclease site within the amplified product in        the absence of a change in one or more nucleotides;    -   digesting the amplified product with the restriction        endonuclease whose site has been potentially introduced in said        amplified product and subjecting single-stranded forms of the        amplified product subjected to hybridization to conditions to        permit annealing to a set of said immobilized oligonucleotides        comprising oligonucleotides which are complementary to a portion        of at least one primer sequence or its complementary sequence;        and    -   detecting the relative proportion of signal by the reporter        molecules wherein an equal proportion of different signals or        the substantial presence of only one signal represents a        homozygous presence or absence of change in the target        nucleotide sequence and wherein the presence of a differential        signal represents a heterozygous presence or absence of said        change in target nucleotide sequence.

Yet another aspect of the present invention further provides an assaydevice for determining the presence or absence of a nucleotide or groupof nucleotides in a nucleic acid molecule comprising an array ofimmobilized oligonucleotides each complementary to a nucleotide sequencewithin an amplified product digested by one or more restrictionendonucleases and means to screen for the hybridization of a targetsequence to the immobilized oligonucleotide array.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatic representation of the genetic assay todetermine the homozygous presence or absence or the presence inheterozygous form of the 35ΔG mutation in the connexin 26 gene.

FIG. 2 is a diagrammatic representation showing (A) wild-type (WT)target sequence for genetic testing for cystic fibrosis; (B) the WTsequence carrying two A→C substitutions creating an XcmI site; (C) a CTTdeletion destroys the XcmI site but creates a BstAI site.

FIG. 3 is a photographic representation showing electrophoreticseparation of amplified products following amplification of DNAputatively encoding a ΔF508 mutation. The target sequence is set forthin<400>2 (Example 4). X, XcmI; B, BstXI; m, marker; N/N, homozygousnormal; ΔF508/ΔF508, homozygous mutation; N/ΔF508, heterozygousmutation.

FIG. 4 is a diagram of a genetic assay to determine the homozygouspresence or absence or the presence in heterozygous form of the 35ΔGmutation in the connexin 26 gene. This is a modified version of themethod described in FIG. 1. The method uses a dually labelled probewhich is annealed to single-stranded DNA as they cleaved.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides inter alia a genetic assay to determinethe homozygous presence or absence of a particular nucleotide orsequence of nucleotides and whether a particular nucleotide ornucleotide sequence is present in heterozygous form.

Accordingly, one aspect of the present invention contemplates a methodfor determining the presence or absence of a homozygous or heterozygouschange in one or more nucleotides within a target nucleotide sequence,said method comprising:—

-   -   amplifying said target nucleotide sequence using forward and        reverse primers to produce an amplified product, wherein at        least one of said primers is labelled with a reporter molecule        capable of facilitating the provision of an identifiable signal        which can be distinguished from another reporter molecule if        both primers are labelled, and wherein at least one primer and        its complementary form comprises a complementary sequence to an        oligonucleotide sequence anchored to a solid support, and        wherein one or more of said forward or reverse primers        introduces, abolishes or hybridizes to a target site within the        amplified product in the presence or absence of a change in one        or more nucleotides, and    -   subjecting said amplified product to detection means.

Preferably, the forward and reverse primers introduce or abolish arestriction endonuclease site.

Accordingly, this aspect of the present invention provides a method fordetermining the presence or absence of a homozygous or heterozygouschange in one or more nucleotides within a target nucleotide sequence,said method comprising:

-   -   amplifying said target nucleotide sequence using forward and        reverse primers to produce an amplified product wherein each of        said primers is labelled with a reporter molecule capable of        facilitating the provision of an identifiable signal which can        be distinguished from each other and wherein at least one primer        and its complementary form comprises a complementary sequence to        an oligonucleotide sequence anchored to a solid support and        wherein one or more said forward or reverse primers introduces        or abolishes a restriction endonuclease site within the        amplified product in the presence or absence of a change in one        or more nucleotides;    -   digesting the amplified product with the restriction        endonuclease whose site has been potentially introduced or        abolished in said amplified product and subjecting        single-stranded forms of the amplified product subjected to        hybridization to conditions to permit annealing to a set of said        immobilized oligonucleotides comprising oligonucleotides which        are sense or complementary to a portion of the amplified        sequence introduced by at least one primer; and    -   detecting the relative proportion of signal by the reporter        molecules wherein an equal proportion of different signals or        the substantial presence of only one signal represents a        homozygous presence or absence of change in the target        nucleotide sequence and wherein the presence of a differential        signal represents a heterozygous presence or absence of said        change in target nucleotide sequence.

In a related embodiment, differential restriction endonuclease digestionmay be determined electrophoretically.

Accordingly, another aspect of the present invention contemplates amethod for determining the presence or absence of a homozygous orheterozygous change in one or more nucleotides within a targetnucleotide sequence, said method comprising:

-   -   amplifying said target nucleotide sequence using forward and        reverse primers to produce an amplified product wherein each of        said primers are labelled with a reporter molecule capable of        facilitating the provision of an identifiable signal which can        be distinguished from each other and wherein one or more of said        forward or reverse primers introduces or abolishes a restriction        endonuclease site within the amplified product in the presence        or absence of a change in one or more nucleotides; and    -   digesting the amplified product with the restriction        endonuclease whose site has been potentially introduced or        abolished in said amplified product and subjecting the digested        amplified product to conditions to permit electrophoretic        separation of said digested products wherein the pattern of        electrophoretic separation and/or the pattern of reporter        molecule signaling is indicative of the homozygous presence or        absence or the heterozygous presence or absence of said change        in target sequence.

In a related embodiment, where electrophoretic separation is employed,the amplification primers are not labelled with a reporter moleculeand/or tag sequence. According to this embodiment, the present inventioncontemplates a method for determining the presence or absence of ahomozygous or heterozygous change in one or more nucleotides within atarget nucleotide sequence such as but not limited to, said methodcomprising:—

-   -   amplifying said target nucleotide sequence using forward and        reverse primers to produce an amplified product wherein one or        more of said forward or reverse primers introduces or abolishes        a restriction endonuclease site within the amplified product in        the presence or absence of a change in one or more nucleotides;        and    -   digesting the amplified product with the restriction        endonuclease whose site has been potentially introduced or        abolished in said amplified product and subjecting the amplified        product subject to digestion to conditions to permit        electrophoretic separation of said digested products wherein the        pattern of electrophoretic separation is indicative of the        homozygous presence or absence or the heterozygous presence or        absence of said change in target sequence.

The present invention contemplates both the introduction of arestriction site or the abolition of a restriction site although theintroduction of a restriction site in, for example, a wild-type or“non-mutation” sequence is preferred.

According to this preferred embodiment, the present invention provides amethod for determining the presence or absence of a homozygous orheterozygous change in one or more nucleotides within a targetnucleotide sequence, said method comprising:

-   -   amplifying said target nucleotide sequence using forward and        reverse primers to produce an amplified product wherein each of        said primers is labelled with a reporter molecule capable of        facilitating the provision of an identifiable signal which can        be distinguished from each other and wherein at least one primer        and its complementary form comprises a complementary sequence to        an oligonucleotide sequence anchored to a solid support and        wherein one or more said forward or reverse primers introduces a        restriction endonuclease site within the amplified product in        the absence of a change in one or more nucleotides;    -   digesting the amplified product with the restriction        endonuclease whose site has been potentially introduced in said        amplified product and subjecting single-stranded forms of the        amplified product subjected to hybridization to conditions to        permit annealing to a set of said immobilized oligonucleotides        comprising oligonucleotides which are complementary to a portion        of each primer sequence or its complementary sequence; and    -   detecting the relative proportion of signal by the reporter        molecules wherein an equal proportion of different signals or        the substantial presence of only one signal represents a        homozygous presence or absence of change in the target        nucleotide sequence and wherein the presence of a differential        signal represents a heterozygous presence or absence of said        change in target nucleotide sequence.

This aspect of the present invention further extends to electrophoreticseparation to determine differential restriction endonuclease digestion.

The restriction site may be introduced or abolished by either theforward primer or the reverse primer. In one particularly usefulembodiment, the forward primer is used to introduce a restriction site.

Accordingly, another aspect of the present invention is directed to amethod for determining the presence or absence of a homozygous orheterozygous change in one or more nucleotides within a targetnucleotide sequence, said method comprising:

-   -   amplifying said target nucleotide sequence using forward and        reverse primers to produce an amplified product wherein said        primers are labelled with a reporter molecule capable of        facilitating the provision of an identifiable signal which can        be distinguished from each other and wherein at least one primer        and its complementary form comprises a complementary sequence to        an oligonucleotide sequence anchored to a solid support and        wherein said forward primer introduces a restriction        endonuclease site within the amplified product in the absence of        a change in one or more nucleotides; digesting the amplified        product with the restriction endonuclease whose site has been        potentially introduced in said amplified product and subjecting        single-stranded forms of the amplified product subjected to        hybridization to conditions to permit annealing to a set of said        immobilized oligonucleotides comprising oligonucleotides which        are complementary to a portion of the at least one primer        sequence or its complementary sequence; and    -   detecting the relative proportion of signal by the reporter        molecules wherein an equal proportion of different signals or        the substantial presence of only one signal represents a        homozygous presence or absence of change in the target        nucleotide sequence and wherein the presence of a differential        signal represents a heterozygous presence or absence of said        change in target nucleotide sequence.

In an alternative embodiment, the target sequence is amplified with anunlabelled set of primers. This is particularly useful forelectrophoretic detection-based assays (e.g. for cystsic fibrosis).Alternatively, again unlabelled primers are used but one of the primerscomprises one or more nucleotides which are chemically modified at thenucleotide or base level or wherein the phosphodiester linkage ismodified so as to provide resistance to an exonuclease. A “chemicallymodified” base or nucleotide includes a nucleotide or base chemicalanalog. One example of a chemical modification is a phosphorothioatemodification or a propyne modification. In essence, the chemicalmodification encompasses any modification which substantially inhibitsthe function of an exonuclease. In a particularly preferred embodiment,the chemical modification is a phosphorothioate modification.

The primers are also selected such that one or other of the primersintroduce or abolish a restriction endonuclease site as described above.Furthermore, as above, at least one of the primers carries a sequence ofnucleotides having a sense or complementary sequence in anoligonucleotide immobilized to a solid support.

After amplification, the resulting amplicon is subjected to exonucleasedigestion. A DNA strand comprising the primer with one or morechemically modified nucleotide bases or phosphodiester linkages isgenerally immune from exonuclease cleavage. Accordingly, the exonucleasedigests only the complementary strand leaving a single-stranded DNAcomprising an introduced or abolished restriction site and a nucleotidesequence having a sense or complementary sequence in an oligonucleotideimmobilized to a solid support. The single-stranded nucleotide sequenceis then contacted by a nucleotide probe that contains complementarity tothe restriction site that may have been introduced. The probe comprisestwo reporter molecules, preferably at its 5′ and 3′ ends and hybridizesto a region encompassing the introduced or abolished restrictionendonuclease site. This hybridization results in a partialdouble-stranded molecule. This molecule is then subjected to digestionconditions. Depending on whether or not the restriction endonucleasesite has been abolished will dictate whether or not the probe iscleaved. One aspect of this method is described in FIG. 4.

Accordingly, another aspect of the present invention contemplates amethod for determining the presence or absence of a homozygous orheterozygous change in one or more nucleotides within a targetnucleotide sequence, said method comprising:—

-   -   amplifying said target nucleotide sequence using forward and        reverse primers to produce an amplified product wherein one        primer comprises one or more chemically modified nucleotides,        bases or phosphodiester bonds such that a nucleotide strand        which extends from said primer is substantially resistant to        exonuclease activity and wherein the other primer comprises a        nucleotide sequences having sense and complementary sequences        immobilized to a solid support and wherein one or more said        forward or reverse primers introduces or abolishes a restriction        endonuclease site within the amplified product in the presence        or absence of a change in one or more nucleotides;    -   digesting the amplified product with an exonuclease to digest        the strand not amplified by the primer comprising the        exonuclease-resistant nucleotides, bases or phosphodiester        linkages to generate a single-stranded nucleic acid molecule        comprising the potential presence or absence of a restriction        endonuclease site and a nucleotide sequence complementary to an        oligonucleotide sequence immobilized to said solid support;    -   hybridizing to said single-stranded nucleic acid molecule a        probe that contains complementarity to the restriction site that        may have been introduced to generate a partial double-stranded        molecule wherein the probe comprises two reporter molecules        capable of facilitating the provision of identifiable signals        which can be distinguished from each other;    -   digesting the double-stranded molecule with the restriction        endonuclease whose site has been potentially introduced or        abolished in said amplified product and subjecting the digested        molecule to conditions to permit annealing to a set of said        immobilized oligonucleotides comprising oligonucleotides which        are sense or complementary to a portion of the amplified        sequence introduced by at least one primer; and    -   detecting the relative proportion of signal by the reporter        molecules wherein an equal proportion of different signals or        the substantial presence of only one signal represents a        homozygous presence or absence of change in the target        nucleotide sequence and wherein the presence of a differential        signal represents a heterozygous presence or absence of said        change in target nucleotide sequence.

The target nucleotide sequence is generally in a eukaryotic cell such asa mammalian (including a human, primate, livestock animal, laboratorytest animal or companion animal cell) or plant cell. In one particularlyuseful embodiment, the target nucleotide sequence is in a human cell.Furthermore, the target nucleotide sequence generally encompasses anucleotide sequence of, for example, a structural gene or regulatorygene or 3′ or 5′ regulatory nucleotide sequences or promoter sequenceswhich are associated with a particular disease condition. Diseaseconditions encompassed by this aspect of the present invention includebut are not limited to disease conditions associated with one or moremutations in one gene or genetic sequence or in a number of known genesor genetic sequences. Examples of disease conditions contemplated hereinfor detection include metabolic disorders such as adreno-leukodystrophy,atherosclerosis, gaucher disease, gyrate atrophy, juvenile onsetdiabetes, obesity, paroxysmal nocturnal hemoglobinuria, phenylketonuria,refsum disease, tangler disease and haemochromatosis conditionsinvolving transporters, channels and pumps such as cystic fibrosis,deafniess, diastrophic dysplasia, long-QT syndrome, Menkes syndrome,Pendred syndrome, polycystic kidney disease, sickle cell anemia,Wilson's disease and Zellweger syndrome, conditions involving signaltransduction such as ataxia telangiectasia, baldness, Cockayne syndrome,glaucoma, tuberous sclerosis, Waardenburg syndrome and Werner syndrome;conditions involving the brain such as Alzheimer's disease, amyotrophiclateral sclerosis, Angleman syndrome, Charcot-Marie-Tooth disease,epilepsy, essential tremor, fragile X syndrome, Friedreich's ataxia,Huntington's disease, Niemann-Pick disease, Parkinson's disease,Prader-Willi syndrome, Rett syndrome, spinocerebella atrophy andWilliam's syndrome; and conditions involving the skeleton such asDuchenne muscular dystrophy, Ellis-van Creveld syndrome, Marfan syndromeand myotonic dystrophy.

Some of the conditions contemplated herein are associated withaberrations in more than one gene or genetic sequence and, hence, anassay may require the interrogation of a number of genes for potentialchanges in nucleotide sequences associated with a disease condition.

Furthermore, the instant invention extends to detecting mutations andpolymorphisms in a range of animal and plant cells. The presentinvention is particularly useful, for example, in screening forpolymorphic variants in the genome of plants such as during the tissueculture stages of plant propagation. The ability to identify polymorphicvariants in plants such as due to somaclonal variation will preventunnecessary resources being wasted on plants with undesired properties.

A change in nucleotide sequence at the homozygous or heterozygous levelis useful for determining the potential seriousness of the disease andin detecting potential carriers of the disease condition. Preferably,the change affects a single nucleotide such as a nucleotidesubstitution, addition or deletion.

The reporter molecule is any molecule capable of facilitating theprovision of an identifiable signal. Suitable reporter molecules includebut are not limited to chloramphenicol which can be acetylated withradioactive acetate groups, colourless galactosidases which may behydrolyzed by galactosidases to yield coloured products, colourlessglucuronides which may be hydrolyzed by glucuronides to yield colouredproducts and fluorescent products, luciferin which maybe oxidized byluciferase to release photons and green fluorescent protein which may beirradiated by U.V. light to emit photons and to fluoresce. A range ofother enzyme-mediated, fluorescent, chemiluminescent and radioactivemarkers may also be employed. The reporter molecule may, therefore,directly or indirectly provide a signal.

Any restriction endonuclease site may be introduced. Suitable sites arerecognized by the following restriction enzymes: AatI, AatII, AauI,Acc113I, Acc16I, Acc65I, AccB1I, AccB7I, AccBSI, AccI, AccII, AccIII,AceIII, AciI, AclI, AClNI, AcMWI, AcsI, AcyI, AdeI, AfaI, AfeI, AflII,AflIII, AgeI, AhaIII, AhdI, AluI, Alw21I, Alw26I, Alw44I, AlwI, AlwNI,Ama87I, AocI, AorflHI, ApaBI, ApaI, ApaLI, ApoI, AscI, AseI, AsiAI,AsnI, Asp700I, Asp718I, AspEI, AspHI, AspI, AspLEI, AspS9I, AsuC2I,AsuHPI, AsuI, AsuII, AsuNHI, AvaI, AvaII, AvafI, AviII, AvrII, AxyI,BaeI, BalI, BamHI, BanI, BanII, Ban If, BbeI, BbilI, BbrPI, BbsI, BbuI,Bbv12I, BbvCI, BbvI, BbviI, BccI, Bce83I, BcefI, BcgI, BciVI, BclI,BcnI, BcoI, BcuI, BetI, BfaI, BfI, BfmI, BfrI, BglI, Bglu, BinI, BlnI,BipI, Bme18I, BmgI, BmrI, BmyI, BpiI, BpRI, BpmI, Bpu10I, Bpu1102I,Bpu14I, BpuAI, Bsa29I, BsaAI, BsaBI, BsaHI, BsaI, BsaJI, BsaMI, BsaOI,BsaWI, BsaXI, BsbI, Bsc4I, BscBI, BscCI, BscFI, BscGI, BscI, Bse118I,BselI, Bse21I, Bse3DI, Bse8I, BseAI, BseCI, BseDI, BseGI, BseLI, BseMI,BseNI, BsePI, BseRI, BseX3I, BsgI, Bsh12361, Bsh12851, Bsh1365I, BshI,BshNI, BsiBI, BsiCI, BsiEI, BsiHKAI, BsiI, BsiLI, BsiMI, BsiQI, BsiSI,BsiWI, BsiXI, BsiYI, BsiZI, BslI, BsmAI, BsmBI, BsmFI, BsmI, BsoBI,Bsp106I, Bsp119I, Bsp120I, Bsp1286I, Bsp13I, Bsp1407I, Bsp143I,Bsp1431I, Bsp1720I, Bsp19I, Bsp24I, Bsp68I, BspA2I, BspCI, BspDI, BspEI,BspGI, BspHI, BspLI, BspLU11I, BspMI, BspMfI, BspTI, BspXI, BsrBI,BsrBR1, BsrDI, BsrFI, BsrGI, BsrI, BsrSI, BssAI, BssHII, BssKI, BssNAI,BssSI, BssT1I, Bst1107I, Bst2BI, Bst2UI, Bst4CI, Bst71I, Bst981, BstACI,BstAPI, BstBAI, BstBI, BstDEI, BstDSI, BstEII, BstF5I, BstH2I, BstHPI,BstMCI, BstNI, BstNSI, BstOI, BstPI, BstSFI, BstSNI, BstUI, BstX2I,BstXI, BstYI, BstZ171, BstZI, Bsu15I, Bsu36I, Bsu6I, BsuRI, BtgI, BtsI,Cac8I, CauII, CbiI, CciNI, Cem, CfoI, Cfr10I, Cfr13I, Cfr42I, Cfr9I,CfrI, CjeI, CjePI, ClaI, CpoI, Csp45I, Csp6I, CspI, CviJI, CviRI, CvnI,DdeI, DpnI, DpnII, DraI, DraII, DraIII, DrdI, DrdII, DsaI, DseDI, EaeI,EagI, Eaml 1041, Eam1105I, EarI, EciI, Ecl136JI, EclHKI, EciXI, Eco10SI,Eco130I, Eco147I, Eco24I, Eco255I, Eco31I, Eco32I, Eco47I, Eco47HI,Eco52I, Eco57I, Eco64I, Eco72I, Eco81I, Eco88I, Eco91I, EcoICR1, EcoNI,EcoO109I, EcoO65I, EcoRI, EcoRII, EcoRV, EcoTl4I, EcoT22I, EcoT38I,EgeI, EheI, ErhI, Esp1396I, Esp3I, EspI, FauI, FauNDI, FbaI, FinI,Fnu4HI, FnuDII, FokI, FriOI, FseI, Fsp4HI, FspI, GdiII, GsuI, HaeI,HaeII, HaeIII, HaeIV, HapH, HgaI, HgiAI, HgiCI, HgiEI, HgiEII, HgiJII,HhaI, HinlI, Hin2I, Hin4I, Hin6I, HincII, HindIII, HindIII, HinfI,HinP1I, HpaI, HpaiI, HphI, Hsp92I, Hsp921I, HspAI, ItaI, KasI, Kpn2I,KpnI, Ksp22I, Ksp6321, KspAI, KspI, Kzo9I, LspI, MaeI, MaeII, MaeIII,MamI, MbiI, MboI, MboiI, McrI, MfeI, MflL, MIsI, MluL, MluNI, Mly1131,MmeI, MnlI, Mph1103I, MroI, MroNI, MroXI, MscI, MseI, MsiI, Msp17I,MspA1I, MspCI, MspI, MspR9I, MstI, MunI, Mva1269I, MvaI, MvnI, MwoI,NaeI, NarI, NciI, NcoI, NdeI, NdeII, NgoAIV, NgoMIV (previously known asNgoMI), NheI, NlaIII, NlaV, NotI, NruGI, NruI, NsbI, NsiI, NspBH, NspI,NspV, PacI, PaeI, PaeR7I, PagI, PalI, PauI, Pf1108I, Pf1231I, PflFI,PflMI, PinAI, Ple19I, PleI, PmaCI, Pme55I, PmeI, PmlI, Ppu10I, PpuMI,PshAI, PshBI, Psp124BI, Psp1406I, Psp5U, PspAI, PspEI, PspLI, PspN4I,PspOMI, PspPPI, PstI, PvuI, PvuII, RcaI, RleAI, RsaI, RsrII, SacI,SacfI, SalI, SanDI, SapI, Sau3AI, Sau96I, SauI, SbfI, ScaI, SchI, ScrFI,SdaI, SduI, SecI, SexAI, SfaNI, SfcI, SfeI, SfiI, SfoI, Sfr274I,Sft303I, SfuI, SgtI, SgrAI, SimI, SinI, SmaI, SmiI, SmlI, SnaBI, SnaI,SpeI, SphI, SplI, SrfI, Sse8387I, Sse8647I, Sse9I, SseBI, SspBI, SspI,SstI, SstH, StuI, StyI, SunI, SwaI, TaiI, TaqI, TaqiI, TatI, TauI, TfiI,ThaI, Tru1I, Tru9I, TscI, TseI, Tsp45I, Tsp4CI, Tsp509I, TspEI, TspRI,Tth111I, Tth111II, TthHB8I, UbaDI, UbaEI, UbaLI, UbaOI, Van91I, Yha464I,VneI, VspI, XagI, XbaI, XcmI, XhoI, XhoII, XmaCI, XmaI, XmaIll and XmnI,Zsp2I.

The solid support is typically glass or a polymer, such as but notlimited to, cellulose, ceramic material, nitrocellulose, polyacrylamide,nylon, polystyrene and its derivatives, polyvinylidene difluoride(PVDF), methacrylate and its derivatives, polyvinyl chloride orpolypropylene. Nitrocellulose may also be used. Glass is particularlypreferred. A solid support may also be a hybrid such as a nitrocellulosefilm supported on a glass or polymer matrix. Reference to a “hybrid”includes reference to a layered arrangement of two or more glass orpolymer surfaces listed above. The solid support may be in the form of amembrane or tubes, beads, discs or microplates, or any other surfacesuitable for conducting an assay. Binding processes to immobilize themolecules are well-known in the art and generally consist of covalentlybinding (e.g. cross linking) or physically adsorbing the molecules tothe solid substrate.

The term “complementary” refers to the topological capability ormatching together of interacting surfaces of an oligonucleotide probeand its target oligonucleotide, which may be part of a largerpolynucleotide. Thus, the target and its probe can be described ascomplementary, and furthermore, the contact surface characteristics arecomplementary to each other. Complementary includes base complementaritysuch as A is complementary to T or U, and C is complementary to G in thegenetic code. However, this invention also encompasses situations inwhich there is non-traditional base-pairing such as Hoogsteen basepairing which has been identified in certain transfer RNA molecules andpostulated to exist in a triple helix. In the context of the definitionof the term “complementary”, the terms “match” and “mismatch” as usedherein refer to the hybridization potential of paired nucleotides incomplementary nucleic acid strands. Matched nucleotides hybridizeefficiently, such as the classical A-T and G-C base pair mentionedabove. Mismatches are other combinations of nucleotides that hybridizeless efficiently.

The term “oligonucleotide” as used herein refers to a polymer composedof a multiplicity of nucleotide residues (deoxyribonucleotides orribonucleotides, or related structural variants or synthetic analogsthereof) linked via phosphodiester bonds (or related structural variantsor synthetic analogs thereof). Thus, while the term “oligonucleotide”typically refers to a nucleotide polymer in which the nucleotideresidues and linkages between them are naturally occurring, it will beunderstood that the term also includes within its scope various analogsincluding, but not restricted to, peptide nucleic acids (PNAs),phosphoramidates, phosphorothioates, methyl phosphonates, 2-O-methylribonucleic acids, and the like. The exact size of the molecule can varydepending on the particular application. An oligonucleotide is typicallyrather short in length, generally from about 8 to 50 nucleotides,preferably 8 to 30 nucleotides, more preferably from about 10 to 20nucleotides and still more preferably from about 11 to 17 nucleotides,but the term can refer to molecules of any length, although the term“polynucleotide” or “nucleic acid” is typically used for largeoligonucleotides. Oligonucleotides may be prepared using any suitablemethod, such as, for example, the phosphotriester method as described inan article by Narang et al. (Methods Enzymol. 68: 90, 1979) and U.S.Pat. No. 4,356,270. Alternatively, the phosphodiester method asdescribed in Brown et al. (Methods Enzymol. 68: 109, 1979) may be usedfor such preparation. Automated embodiments of the above methods mayalso be used. For example, in one such automated embodiment,diethylphosphoramnidites are used as starting materials and may besynthesized as described by Beaucage et al. (Tetrahedron Letters 22:1859-1862, 1981). Reference also may be made to U.S. Pat. Nos. 4,458,066and 4,500,707, which refer to methods for synthesizing oligonucleotideson a modified solid support. It is also possible to use a primer, whichhas been isolated from a biological source (such as a denatured strandof a restriction endonuclease digest of plasmid or phage DNA). In apreferred embodiment, the oligonucleotide is synthesized according tothe method disclosed in U.S. Pat. No. 5,424,186 (Fodor et al.). Thismethod uses lithographic techniques to synthesize a plurality ofdifferent oligonucleotides at precisely known locations on a substratesurface.

The terms “array” and in particular “DNA array” or “oligonucleotidearray” refer to a substrate having oligonucleotide probes with differentknown sequences deposited at discrete known locations associated withits surface. For example, the substrate can be in the form of atwo-dimensional substrate as described in U.S. Pat. No. 5,424,186. Suchsubstrate may be used to synthesize two-dimensional spatially addressedoligonucleotide (matrix) arrays. Alternatively, the substrate may becharacterized in that it forms a tubular array in which atwo-dimensional planar sheet is rolled into a three-dimensional tubularconfiguration. The substrate may also be in the form of a microsphere orbead connected to the surface of an optic fibre as, for example,disclosed by Chee et al. in WO 00/39587. Oligonucleotide arrays have atleast two different features and a density of at least 400 features percm². In certain embodiments, the arrays can have a density of about 500,at least one thousand, at least 10 thousand, at least 100 thousand, atleast one million or at least 10 million features per cm². For example,as stated above, the substrate may be silicon or glass and can have thethickness of a glass microscope slide or a glass cover slip, or may becomposed of other synthetic polymers. Substrates that are transparent tolight are useful when the method of performing an assay on the substrateinvolves optical detection. The term also refers to a probe array andthe substrate to which it is attached that form part of a wafer.

The term “probe” refers to an oligonucleotide molecule that binds to aspecific target sequence or other moiety of another nucleic acidmolecule. Unless otherwise indicated, the term “probe” in the context ofthe present invention typically refers to an oligonucleotide probe thatbinds to another oligonucleotide or polynucleotide, often called the“target polynucleotide”, through complementary base pairing. Probes canbind target polynucleotides lacking complete sequence complementaritywith the probe, depending on the stringency of the hybridizationconditions.

Oligonucleotide probes may be selected to be “substantiallycomplementary” to a target sequence as defined herein. The exact lengthof the oligonucleotide probe will depend on many factors includingtemperature and source of probe and use of the method. For example,depending upon the complexity of the target sequence, theoligonucleotide probe may typically contain 8 to 50 nucleotides,preferably 8 to 30 nucleotides, more preferably from about 10 to 20nucleotides and still more preferably from about 11 to 17 nucleotidescapable of hybridization to a target sequence although it may containmore or fewer such nucleotides.

The term “similarity” as used herein includes exact identity betweencompared sequences at the nucleotide or amino acid level. Where there isnon-identity at the nucleotide level, “similarity” includes differencesbetween sequences which result in different amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. Where there is non-identity atthe amino acid level, “similarity” includes amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. In a particularly preferredembodiment, nucleotide and sequence comparisons are made at the level ofidentity rather than similarity.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence”,“comparison window”, “sequence similarity”, “sequence identity”,“percentage of sequence similarity”, “percentage of sequence identity”,“substantially similar” and “substantial identity”. A “referencesequence” is at least 12 but frequently 15 to 18 and often at least 25or above, such as 30 monomer units, inclusive of nucleotides and aminoacid residues, in length. Because two polynucleotides may each comprise(1) a sequence (i.e. only a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) asequence that is divergent between the two polynucleotides, sequencecomparisons between two (or more) polynucleotides are typicallyperformed by comparing sequences of the two polynucleotides over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window” refers to a conceptual segment oftypically 12 contiguous residues that is compared to a referencesequence. The comparison window may comprise additions or deletions(i.e. gaps) of about 20% or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by computerized implementations ofalgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, 575 Science DriveMadison, Wis., USA) or by inspection and the best alignment (i.e.resulting in the highest percentage homology over the comparison window)generated by any of the various methods selected. Reference also may bemade to the BLAST family of programs as, for example, disclosed byAltschul et al. (Nucl. Acids Res. 25: 3389. 1997). A detailed discussionof sequence analysis can be found in Unit 19.3 of Ausubel et al.(“Current Protocols in Molecular Biology” John Wiley & Sons Inc,1994-1998, Chapter 15.).

The terms “sequence similarity” and “sequence identity” as used hereinrefers to the extent that sequences are identical or functionally orstructurally similar on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity”, for example, is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala,Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp,Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. For the purposes of the present invention, “sequenceidentity” will be understood to mean the “match percentage” calculatedby the DNASIS computer program (Version 2.5 for windows; available fromHitachi Software engineering Co., Ltd., South San Francisco, Calif.,USA) using standard defaults as used in the reference manualaccompanying the software. Similar comments apply in relation tosequence similarity.

Reference herein to a low stringency includes and encompasses from atleast about 0 to at least about 15% v/v formamide and from at leastabout 1 M to at least about 2 M salt for hybridization, and at leastabout 1 M to at least about 2 M salt for washing conditions. Generally,low stringency is at from about 25-30° C. to about 42° C. Thetemperature may be altered and higher temperatures used to replaceformamide and/or to give alternative stringency conditions. Alternativestringency conditions may be applied where necessary, such as mediumstringency, which includes and encompasses from at least about 16% v/vto at least about 30% v/v formamide and from at least about 0.5 M to atleast about 0.9 M salt for hybridization, and at least about 0.5 M to atleast about 0.9 M salt for washing conditions, or high stringency, whichincludes and encompasses from at least about 31% v/v to at least about50% v/v formamide and from at least about 0.01 M to at least about 0.15M salt for hybridization, and at least about 0.01 M to at least about0.15 M salt for washing conditions. In general, washing is carried outT_(m)=69.3+0.41 (G+C)% (Marmur and Doty, J. Mol. Biol. 5: 109, 1962).However, the T_(m) of a duplex DNA decreases by 1° C. with everyincrease of 1% in the number of mismatch base pairs (Bonner and Laskey,Eur. J. Biochem. 46: 83, 1974). Formamide is optional in thesehybridization conditions. Accordingly, particularly preferred levels ofstringency are defined as follows: low stringency is 6×SSC buffer, 0.1%w/v SDS at 25-42° C.; a moderate stringency is 2×SSC buffer, 0.1% w/vSDS at a temperature in the range 20° C. to 65° C.; high stringency is0.1×SSC buffer, 0.1% w/v SDS at a temperature of at least 65° C.

The terms “target polynucleotide” or “target sequence” refer to apolynucleotide of interest (e.g. a single gene or polynucleotide) or agroup of polynucleotides (e.g. a family of polynucleotides). The targetpolynucleotide can designate mRNA, RNA, eRNA, cDNA or DNA. The probe isused to obtain information about the target polynucleotide: whether thetarget polynucleotide has affinity for a given probe. Targetpolynucleotides may be naturally occurring or man-made nucleic acidmolecules. Also, they can be employed in their unaltered state or asaggregates with other species. Target polynucleotides may be associatedcovalently or non-covalently, to a binding member, either directly orvia a specific binding substance. A target polynucleotide can hybridizeto a probe whose sequence is at least partially complementary to asub-sequence of the target polynucleotide.

These terms are also used herein to refer to a chosen nucleotidesequence of at most 300, 250, 200, 150, 100, 75, 50, 30, 25 or at most15 nucleotides in length. Target sequences include sequences of at least8, 10, 15, 25, 30, 35, 45, 50, 60, 70, 80, 90, 100, 120, 135, 150, 175,200, 250 and 300 nucleotides in length. Non-limiting examples of targetsequences include, but are not restricted to, repeat sequences such asAlu repeat sequences, conserved or non-conserved regions of genefamilies, introns, promoter sequences including the Hogness Box and theTATA box, signal sequences, enhancers, protein-binding domains such as ahomeobox, tymobox, polymorphisms and conserved protein domains orportions thereof.

Hybridization and/or reporter signal data are processed to determine thepresence or absence of a restriction endonuclease site. In a preferredembodiment, a digital computer is employed to correlate specificpositional labelling on the array with the presence of any of the targetsequences for which the probes have specificity of interaction. Thepositional information is directly converted to a database indicatingwhat sequence interactions have occurred. Data generated inhybridization assays are most easily analyzed with the use of aprogrammable digital computer. The computer program product generallycontains a readable medium that stores the codes. Certain files aredevoted to memory that includes the location of each feature and all thetarget sequences known to contain the sequence of the oligonucleotideprobe at that feature. Computer methods for analyzing hybridization datafrom nucleic acid arrays is taught in International Patent PublicationNo WO 97/29212 and EP Publication 95307476.2. In a preferred embodiment,the prograrrmable computer would contain specialist software code andregister data derived from the entire sequence database, or containingthat part of the entire sub-sequence database that is relevant to theparticular probe array, and from the pattern of hybridization willassess the probability that particular target sequences were present inthe tested DNA sample.

The computer program product can also contain code that receives asinput hybridization data from a hybridization reaction between a targetsequence and an oligonucleotide probe. The computer program product canalso include code that processes the hybridization data.

Data analysis can include the steps of determining, for example, thefluorescence intensity as a function of substrate position from the datacollected, removing “outliers” (data deviating from a predeterminedstatistical distribution) and calculating the relative binding affinityof the target sequences from the remaining data. The resulting data canbe displayed as an image with colour in each region varying according tothe light emission or binding affinity between target sequences andprobes therein.

In one embodiment, the amount of binding at each address is determinedby examining the on-off rates of the hybridization. For example, theamount of binding at each address is determined at several time pointsafter the nucleic acid sample is contacted with the array. The amount oftotal hybridization can be determined as a function of the kinetics ofbinding based on the amount of binding at each time point.

Persons of skill in the art can easily determine the dependence of thehybridization rate on temperature, sample agitation, washing conditions(e.g. pH, solvent characteristics, temperature) in order to maximiseconditions for hybridisztion rate and signal to noise.

The computer program product also can include code that receivesinstructions from a programmer as input. The computer program productmay also transform the data into a format for presentation.

In one embodiment, the computer program product for processinghybridization data comprises code that identifies for each targetpolynucleotide a combination of features in an oligonucleotide arraywhose probes facilitate specific detection of that polynucleotide; codethat receives as input hybridization data from hybridization reactionsbetween sample polynucleotides and the oligonucleotide probes in thearray; code that processes the hybridisation data to determine whetherthe sample polynucleotides comprises any of the target polynucleotidesby searching for hybridization patterns that match any of the predefinedcombinations of target sequences; codes that identify the presence of areporter molecule-mediated signal; and a computer readable medium thatstores the codes. It is not necessary to identify the sequence ofrespective oligonucleotide probes in each feature of the array. In thisrespect, the hybridization analysis software only requires as inputwhich combination of features in the array corresponds to a particulartarget polynucleotide. However, in a preferred embodiment, the computerprogram product comprises code that receives as input the sequence of anoligonucleotide probe in each feature of an oligonucleotide array andcode that receives as input a database that contains information on thepresence or absence of target sequences in target polynucleotides.

Preferably the computer program product further comprises code thatdeduces the probability that the detected pattern of hybridizationindicates the presence of a target polynucleotide.

The database of target sequences would be regularly up-dated and thepart of it relevant to each particular set of probes forming eachmicro-array would also be updated for those using particular commercialapplications of the invention.

The method of the present invention may also be modified to introduceone particular restriction endonuclease site but to abolish anothersite. This provides even more accuracy and a reduced likelihood of afalse negative or false positive. In addition, it is not necessary thata primer has to introduce a restriction endonuclease site. A particularsite may be naturally present in a target sequence.

The present invention further provides an assay device for determiningthe presence or absence of a nucleotide or group of nucleotides in anucleic acid molecule comprising an array of immobilizedoligonucleotides each complementary to a nucleotide sequence within anamplified product digested by one or more restriction endonucleases andmeans to screen for the hybridization of a target sequence to theimmobilized oligonucleotide array. The assay device may also be packagedfor sale and contain instructions for use.

The present invention is further described by the following non-limitingExamples.

EXAMPLE 1 Development of Genetic Deafness Assay: EcoRII Assay

In this assay, a mutation at nucleotide 35 in the connexin 26 gene isidentified either in the homozygous or heterozygous state. The mutationis a deletion of a guanine at position 35. This mutation is referred toas “35 ΔG”.

Two primers are developed, each labelled with a different reportermolecule and at least one comprising a nucleotide sequence matching andcomplementary to oligonucleotide sequences immobilized to a solidsupport. This sequence on the primer is referred to as a “tag” sequence.Conveniently, in one example, the GeneChip (registered trademark) isused incorporating GenFlex (trademark) Tag array. The primers comprise,therefore, a reporter molecule alone or linked to a tag sequence (havingmatching and complementary sequences immobilized to a solid support). Inthis example, one primer comprises a tag sequence linked to a nucleotidesequence complementary to a region flanking the 35ΔG region for theforward primer and a region downstream of this location for the reverseprimer. In one example, the reverse primer introduces a base change inthe wild-type sequence thus creating a EcoRII site. If the targetsequence comprises a 35ΔG mutation then the EcoRII site is lost. This isbecause EcoRII recognizes the nucleotide sequence 5′CCWGG3′ where W is Aor T. In the connexin 26 gene, the nucleotide sequence recognized byEcoRII is 5′CCTGG3′. However, a 35ΔG mutation removes the G at the 3′position and, hence, amplification product from a 35ΔG sample will notdigest but a wild-type sequence will digest. After amplification anddigestion with EcoRII, single-stranded forms of the amplified productare exposed to the immobilized oligonucleotides on the solid support.This assay is shown in FIG. 1.

As can be observed, when the target sequence is homozygous wild-type,all the amplification product will be digested thus removing thereporter molecule associated with the reverse primer. The complementary(antisense) immobilized oligonucleotide (+) permits capture of the tagassociated with the forward primer. The matching (sense) immobilizedoligonucleotide (−) permits capture of the sequence complementary to thetag generated by extension of the reverse primer during PCR (ie thecomplementary strand to the strand generated by extension of the forwardprimer). Regardless of restriction enzyme digestion, the reportermolecule associated with the forward primer will always be detected atthe (+) feature of the immobilized oligonucleotide pair specific for thetag associated with that forward primer. As the reporter moleculeassociated with the reverse primer has been cleaved away by EcoRIIdigestion, no reporter molecule will be detected at the (−) feature ofthe immobilized oligonucleotide pair, i.e. the ratio of signal fromforward to reverse primer would be in the order of 1:0.

If the 35ΔG mutation is present in a homozygous state, there will be nodigestion of any amplification product and both reporter molecules onboth primers will be equally represented, i.e. in a ratio of about 1:1.

If the 35ΔG mutation is in the heterozygous state, then theamplification product from the nucleotide sequence carrying the mutationwill not cleave but cleavage will occur in the amplification productfrom the nicleotide sequence not carrying the mutation. Therefore, abouthalf of the molecules in the total amplification will be cleaved.Accordingly, the ratio of signal from forward to reverse primer will beapproximately 1:0.5.

EXAMPLE 2 Development of Genetic Deafness Assay: DdeI Assay

In this assay, the same approach as adopted in Example 1 but a mutationis introduced to create a DdeI site in the 35ΔG sequence. A targetvariant would, therefore, be cleaved by DdeI, whereas the wild-typesequence would not.

EXAMPLE 3 Development of Genetic Deafness Assay: BclI Assay

The aim of this assay is to use a primer to change a cytosine to anadenine thus creating a BclI site in the Cx26 gene. The targetnucleotide sequence is as follows:—

-   -   CGC ATT ATG ATC CTC GTT GTG (SEQ ID NO:1).

A reverse primer creates a mismatch such that the TGATCC sequencebecomes TGATCA which is the recognition sequence for Bcll. Wild-typeamplification product based on this modified sequence is digestible byBcli. A mutation in the ATG codon leading to genetic deafness results inthe codon changing to ACG (i.e. a T→C substitution).This corresponds toan M34T substitution. The Bcll site, i.e. TGATCA, becomes CGATCA and,hence, amplification product carrying this mutation is no longerdigestible by Bcll.

EXAMPLE 4 Development of Assay for Cystic Fibrosis

This assay is predicated on enzyme recognition sequences for Xcml andBstX1. The cystic fibrosis gene comprises the following targetsequence:—

-   -   5′AAA GAA AAT ATC ATC TTT GGT GTT TCC TA (SEQ ID NO:2).

Mutations giving rise to a potential development of cystic fibrosisinclude a deletion of a phenylalanine residue at position 508, i.e.ΔF508. This results from a deletion of a CTT codon.

The first step is to use a reverse primer to introduce two A→Csubstitutions to create the XcmI site: CCA(N)⁹TGG.

As a result, wild-type amplification product is digested by XcmI. If theCTT codon is deleted (see FIG. 2), then XcmI does not digest theamplified DNA.

BstX1 has the recognition sequence CCA(N)⁶TGG. BstX1 does not digest thewild-type sequence (FIG. 2). A CTT deletion and the A→C substitutionscreates a Bstm site. Accordingly, where the CTT deletion has occurred,the amplification product is XcmI ^(−ve)/BstX1^(+ve) whereas thewild-type is XcmI^(+ve)/BstX1^(−ve).

This assay may be conducted using the solid array technology as inExamples 1-3 or may be used in conjunction with electrophoreticseparation. The amplification primer sequences need not be labelled witha reporter molecule and/or a tag sequence. An example of electrophoreticseparation is shown in FIG. 3. The differential restriction pattern canbe seen between homozygous normal (NI/N), homozygous abnormal(ΔF508/ΔF508) and heterozygous normal (N/ΔF508).

EXAMPLE 5 Combination Assay for Cystic Fibrosis

A combination assay is conducted using biochemical and genetic testing.

In the combination assay, all babies are subjected to a biochemical testfor cystic fibrosis. Where there is no biochemical indication of amutation, the baby is placed in a non-risk category. If the biochemicaltest suggests the presence of a mutation, then a genetic test isconducted such as outlined in Example 4. Gel electrophoresis (such aspolyacrylamide or agarose gel electrophoresis) is carried out using oneor both XcmI and/or BstXI or an array technology may be employed.

EXAMPLE 6 Development of Chip Technology

Table 1 provides a list of 15 tags which are used in conjunction withPCR oligos. Table 2 is a list of chip probes. These are sense andantisense capture probes which are immobilized to the array.

TABLE 1 Chip TAG list  1 ProbeSet01548 GCTGCTCGTGGTTAAGCTCT High [SEQ IDNO: 3]  2 ProbeSet00138 CGTACCAATGGATGCGGTCT High [SEQ ID NO: 4]  3ProbeSet00357 GAGGTCAGTTCACGAAGCTC High [SEQ ID NO: 5]  4 ProbeSet00468GAGTTCCCGTGCGTTAGATC High [SEQ ID NO: 6]  5 ProbeSet00512GCGACTAGGTGGCTCTAATC High [SEQ ID NO: 7]  6 ProbeSet01873AGTCAAGCTAGATGCCGATC High [SEQ ID NO: 8]  7 ProbeSet00007AAACCATCGACTCACGGGAT High [SEQ ID NO: 9]  8 ProbeSet00156ATGCAGCGTAGGTATCGACT High [SEQ ID NO: 10]  9 ProbeSet01052TACAACGATTGCCTGCCTGT High [SEQ ID NO: 11] 10 ProbeSet01113CACAGAGCTGAGTCGGATAT High [SEQ ID NO: 12] 11 ProbeSet01820TCAGCGCGTGTCGTTGCATA High [SEQ ID NO: 13] 12 ProbeSet01253TTGAATCGTTTGAATCGCGG High [SEQ ID NO: 14] 13 ProbeSet01814CATGCAGCTCGATCTAGCGA High [SEQ ID NO: 15] 14 ProbeSet01790CATGCAGCTCGATCTAGCGA High [SEQ ID NO: 16] 15 ProbeSet01081CTTGATACGACTGTCATGGC High [SEQ ID NO: 17] HC ProbeSet00661CTTGATACGACTGTCATGGC High [SEQ ID NO: 18]

TABLE 2 Chip probe sets 5′→3′  1+ AGAGCTTAACCACGAGCAGC [SEQ ID NO: 19] 1− CGTACCAATGGATGCGGTCT [SEQ ID NO: 20]  2+ AGACCGCATCCATTGGTACG [SEQID NO: 21]  2− CGTACCAATGGATGCGGTCT [SEQ ID NO: 22]  3+GAGCTTCGTGAACTGACCTC [SEQ ID NO: 23]  3− GAGGTCAGTTCACGAAGCTC [SEQ IDNO: 24]  4+ GATCTAACGCACGGGAACTC [SEQ ID NO: 25]  4−GAGTTCCCGTGCGTTAGATC [SEQ ID NO: 26]  5+ GATTAGAGCCACCTAGTCGC [SEQ IDNO: 27]  5− GCGACTAGGTGGCTCTAATC [SEQ ID NO: 28]  6+GATCGGCATCTAGCTTGACT [SEQ ID NO: 29]  6− AGTCAAGCTAGATGCCGATC [SEQ IDNO: 30]  7+ ATCCCGTGAGTCGATGGTTT [SEQ ID NO: 31]  7−AAACCATCGACTCACGGGAT [SEQ ID NO: 32]  8+ AGTCGATACCTACGCTGCAT [SEQ IDNO: 33]  8− ATGCAGCGTAGGTATCGACT [SEQ ID NO: 34]  9+ACAGGCAGGCAATCGTTGTA [SEQ ID NO: 35]  9− TACAACGATTGCCTGCCTGT [SEQ IDNO: 36] 10+ ATATCCGACTCAGCTCTGTG [SEQ ID NO: 37] 10−CACAGAGCTGAGTCGGATAT [SEQ ID NO: 38] 11+ TATGCAACGACACGCGCTGA [SEQ IDNO: 39] 11− TCAGCGCGTGTCGTTGCATA [SEQ ID NO: 40] 12+CCGCGATTCAAACGATTCAA [SEQ ID NO: 41] 12− TTGAATCGTTTGAATCGCGG [SEQ IDNO: 42] 13+ TCGCTAGATCGAGCTGCATG [SEQ ID NO: 43] 13−CATGCAGCTCGATCTAGCGA [SEQ ID NO: 44] 14+ TGTGCGATCCTACTGACCGT [SEQ IDNO: 45] 14− ACGGTCAGTAGGATCGCACA [SEQ ID NO: 46] 15+GCCATGACAGTCGTATCAAG [SEQ ID NO: 47] 15− CTTGATACGACTGTCATGGC [SEQ IDNO: 48] HC− ACGCAGTGAGTAGCATCCTG [SEQ ID NO: 49]

EXAMPLE 7 Development of Assay Involving Dual Labelled Probes

FIG. 4 is a diagrammatic representation of a modified assay involving adual labelled oligonucleotide primer. The dual lableled primer isconnected to single-stranded DNA and then cleaved.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

1. A method for determining the presence or absence of a homozygous orheterozygous mutation in a gene, said method comprising: amplifying atarget nucleotide sequence within the gene using a first and a secondprimer to produce an amplified product, wherein said first primercomprises one or more chemically modified nucleotides, bases orphosphodiester bonds such that a nucleotide strand which extends fromsaid first primer is substantially resistant to exonuclease activity,and wherein said second primer comprises a nucleotide sequence whichintroduces in the strand extended by said first primer a tag sequencewhich is complementary to a capture oligonucleotide immobilized to asolid support and wherein one or both of said first or second primersintroduces a restriction site in the target sequence when there is nomutation; digesting the amplified product with an exonuclease to digestthe strand not amplified by the primer comprising theexonuclease-resistant nucleotides, bases or phosphodiester linkages togenerate a single-stranded nucleic acid molecule; hybridizing to saidsingle-stranded nucleic acid molecule a probe that containscomplementarity to a region encompassing the restriction site that isintroduced when no mutation is present to generate a partialdouble-stranded molecule wherein the probe comprises two reportermolecules capable of facilitating the provision of identifiable signalswhich can be distinguished from each other; contacting the partiallydouble-stranded molecules with a restriction endonuclease which willdigest the double-stranded molecules which do not contain the mutationand subjecting digested and/or non-digested molecules to conditions topermit annealing of the tag sequence to the capture oligonucleotideimmobilized to the solid support; measuring the relative intensity ofthe signal by the reporter molecules; wherein an equal intensity ofdifferent signals represents a homozygous presence of the mutation;wherein one signal represents an absence of the mutation; and wherein amixture of signals, in which the intensity of one signal is less thanthe intensity of the other signal thereby represents a heterozygouspresence of the mutation.
 2. A method for determining the presence orabsence of a homozygous or heterozygous mutation in a gene, said methodcomprising: amplifying a target nucleotide sequence within the geneusing a first and second primer to produce an amplified product, whereinsaid first primer comprises one or more chemically modified nucleotides,bases or phosphodiester bonds such that a nucleotide strand whichextends from said first primer is substantially resistant to exonucleaseactivity, and wherein said second primer comprises a nucleotide sequencewhich introduces in the strand extended by said first primer a tagsequence which is complementary to a capture oligonucleotide immobilizedto a solid support and wherein one or both of said first or secondprimers removes a restriction site in the target sequence when there isno mutation; digesting the amplified product with an exonuclease todigest the strand not amplified by the primer comprising theexonuclease-resistant nucleotides, bases or phosphodiester linkages togenerate a single-stranded nucleic acid molecule; hybridizing to saidsingle-stranded nucleic acid molecule a probe that containscomplementarity to a region encompassing the restriction site that isremoved when no mutation is present to generate a partialdouble-stranded molecule wherein the probe comprises two reportermolecules capable of facilitating the provision of identifiable signalswhich can be distinguished from each other; contacting the partiallydouble-stranded molecules with a restriction endonuclease which willdigest the double-stranded molecules which contain the mutation andsubjecting digested and/or non-digested molecules to conditions topermit annealing of the tag sequence to the capture oligonucleotideimmobilized to the solid support; measuring the relative intensity ofthe signal by the reporter molecules wherein an equal intensity ofdifferent signals represents a homozygous absence of the mutation;wherein one signal represents a homozygous mutation; and wherein amixture of signals, in which the intensity of one signal is less thanthe intensity of the other signal thereby represents a heterozygouspresence of the mutation.
 3. A method of claim 1 or 2 wherein the solidsupport is selected from the list comprising glass and a polymer such ascellulose, nitrocellulose, ceramic material, polyacrylamide, nylon,polystyrene and its derivatives, polyvinylidene difluoride, methacrylateand its derivatives, polyvinyl chloride and polypropylene.
 4. A methodof claim 3 wherein the solid support is glass.
 5. A method of any one ofclaims 1 to 2 wherein two or more oligonucleotide sequences are anchoredto the solid support in the form of an array.
 6. A method any one ofclaims 1 to 2 wherein the restriction endonuclease site is recognized bya restriction enzyme selected from the list comprising AatI, AatII,AauI, Acc113I, Acc16I, Acc65I, AccB1I, AccB7I, AccBSI, AccI, AccII,AccIII, AceIII, AciI, AclI, AclNI, AclWI, AcsI, AcyI, AdeI, AfaI, AfeI,AflII, AflIII, AgeI, AhaIII, AhdI, AluI, Alw21I, Alw26I, Alw44I, AlwI,AlwNI, Ama87I, AocI, Aor51HI, ApaBI, ApaI, ApaLI, ApoI, AscI, AseI,AsiAI, AsnI, Asp700I, Asp718I, AspEI, AspHI, AspI, AspLEI, AspS9I,AsuC2I, AsuHPI, AsuI, AsuII, AsuNHI, AvaI, AvaII, AvaIII, AviII, AvrII,AxyI, BaeI, BalI, BamHI, BanI, BanII, BanIII, BbeI, BbiII, BbrPI, BbsI,BbuI, Bbv12I, BbvCI, BbvI, BbvII, BccI, Bce83I, BcefI, BcgI, BciVI,BclI, BcnI, BcoI, BcuI, BetI, BfaI, BfiI, BfmI, BfrI, BglI, BglII, BinI,BlnI, BlpI, Bme18I, BmgI, BmrI, BmyI, BpiI, BplI, BpmI, Bpu10I,Bpu1102I, Bpu14I, BpuAI, Bsa29I, BsaAI, BsaBI, BsaHI, BsaI, BsaJI,BsaMI, BsaOI, BsaWI, BsaXI, BsbI, Bsc4I, BscBI, BscCI, BscFI, BscGI,BscI, Bse118I, Bse1I, Bse21I, Bse3DI, Bse8I, BseAI, BseCI, BseDI, BseGI,BseLI, BseMII, BseNI, BsePI, BseRI, BseX3I, BsgI, Bsh1236I, Bsh1285I,Bsh1365I, BshI, BshNI, BsiBI, BsiCI, BsiEI, BsiHKAI, BsiI, BsiLI, BsiMI,BsiQI, BsiSI, BsiWI, BsiXI, BsiYI, BsiZI, BslI, BsmAI, BsmBI, BsmFI,BsmI, BsoBI, Bsp106I, Bsp119I, Bsp120I, Bsp1286I, Bsp13I, Bsp1407I,Bsp143I, Bsp143II, Bsp1720I, Bsp19I, Bsp24I, Bsp68I, BspA2I, BspCI,BspDI, BspEI, BspGI, BspHI, BspLI, BspLU11I, BspMI, BspMII, BspTI,BspXI, BsrBI, BsrBRI, BsrDI, BsrFI, BsrGI, BsrI, BsrSI, BssAI, BssHII,BssKI, BssNAI, BssSI, BssT1I, Bst1107I, Bst2BI, Bst2UI, Bst4CI, Bst71I,Bst98I, BstACI, BstAPI, BstBAI, BstBI, BstDEI, BstDSI, BstEII, BstF5I,BstH2I, BstHPI, BstMCI, BstNI, BstNSI, BstOI, BstPI, BstSFI, BstSNI,BstUI, BstX2I, BstXI, BstYI, BstZ17, BstZI, Bsu15I, Bsu36I, Bsu6I,BsuRI, BtgI, BtsI, Cac8I, CauII, CbiI, CciNI, CelII, CfoI, Cfr10I,Cfr13I, Cfr42I, Cfr9I, CfrI, CjeI, CjePI, ClaI, CpoI, Csp45I, Csp6I,CspI, CviJI, CviRI, CvnI, DdeI, DpnI, DpnII, DraI, DraII, DraIII, DrdI,DrdII, DsaI, DseDI, EaeI, EagI, Eam1104I, Eaml 1105I, EarI, EciI,Ec136II, EclHKI, EclXI, Eco105I, Eco130I, Eco147I, Eco24I, Eco255I,Eco31I, Eco32I, Eco47I, Eco47III, Eco52I, Eco57I, Eco64I, Eco72I,Eco81I, Eco88I, Eco91I, EcoICRI, EcoNI, EcoO109I, EcoO65I, EcoRI,EcoRII, EcoRV, EcoT14I, EcoT22I, EcoT38I, EgeI, EheI, ErhI, Esp1396I,Esp3I, EspI, FauI, FauNDI, FbaI, FinI, Fnu4HI, FnuDUII, FokI, FriOI,FseI, Fsp4HI, FspI, GdiII, GsuI, HaeI, HaeII, HaeIII, HaeIV, HapII,HgaI, HgiAI, HgiCI, HgiEI, HgiEII, HgiJII, HhaI, Hin1I, Hin2I, Hin4I,Hin6I, HincII, HindII, HindIII, HinfI, HinP1I, HpaI, HpaII, HphI,Hsp92I, Hsp92II, HspAI, ItaI, KasI, Kpn2I, KpnI, Ksp22I, Ksp632I, KspAI,KspI, Kzo9I, LspI, MaeI, MaeII, MaeIII, MamI, MbiI, MboI, MboII, McrI,MfeI, MflI, MlsI, MluI, MluNI, Mly113I, MmeI, MnlI, Mph1103I, MroI,MroNI, MroXI, MscI, MseI, MslI, Msp171, MspA1I, MspCI, MspI, MspR9I,MstI, MunI, Mva1269I, MvaI, MvnI, MwoI, NaeI, NarI, NciI, NcoI, NdeI,NdeII, NgoAIV, NgoMIV (previously known as NgoMI), NheI, NlaIII, NlaIV,NotI, NruGI, NruI, NsbI, NsiI, NspBII, NspI, NspV, PacI, PaeI, PaeR7I,PagI, PalI, PauI, Pfl1108I, Pfl23II, PflFI, PflMI, PinAI, Ple19I, PleI,PmaCI, Pme55I, PmeI, PmlI, Ppu10I, PpuMI, PshAI, PshBI, Psp124BI,Psp1406I, Psp5II, PspAI, PspEI, PspLI, PspN4I, PspOMI, PspPPI, PstI,PvuI, PvuII, RcaI, RleAI, RsaI, RsrII, SacI, SacII, SalI, SanDI, SapI,Sau3AI, Sau96I, SauI, SbfI, ScaI, SchI, ScrFI, SdaI, SduI, SecI, SexAI,SfaNI, SfcI, SfeI, SfiI, SfoI, Sfr274I, Sfr303I, SfuI, SgfI, SgrAI,SimI, SinI, SmaI, SmiI, SmlI, SnaBI, SnaI, SpeI, SphI, SplI, SrfI,Sse8387I, Sse8647I, Sse9I, SseBI, SspBI, SspI, SstI, SstII, StuI, StyI,SunI, SwaI, TaiI, TaqI, TaqII, TatI, TauI, TfiI, ThaI, TruII, Tru9I,TscI, TseI, Tsp45I, Tsp4CI, Tsp509I, TspEI, TspRI, Tth111I, Tth111II,TthHB8I, UbaDI, UbaEI, UbaLI, UbaOI, Van91I, Vha4641, VneI, VspI; XagI,XbaI, XcmI, XhoI, XhoII, XmaCI, XmaI, XmaIII and XmnI, Zsp2I.
 7. Amethod of any one of claims 1 to 2 wherein the reporter molecule isselected form the list comprising chloramphenicol, a colourlessgalactosidase, a colourless glucuronide, luciferin and green fluorescentprotein.
 8. A method of any one of claims 1 to 2 wherein the targetnucleotide sequence is in a eukaryotic cell.
 9. A method of claim 8wherein the cell is a mammalian cell.
 10. A method of claim 9 whereinthe mammalian cell comprises a target sequence, wherein a mutation isassociated with a disease condition.